Early environmental stress can permanently reprogram hormonal, physiological and behavioral systems. In the neonatal intensive care unit (NICU) preterm infants born at very low gestational age (d32 weeks gestation) are exposed to prolonged pain and stress related to repeated procedures. Our current work is the first to show that: 1) re-programming of stress systems occurs in preterm infants; 2) higher cumulative neonatal stress/procedural pain (independent of early illness severity and intravenous morphine exposure) predicts (a) downregulation of cortisol in the NICU while the infants are exposed to ongoing stressors, but upregulation later in infancy, and (b) poorer cognitive and motor development; 3) the overall allostatic load in preterm infants in the NICU is substantial, since stress of noninvasive clustered nursing care induces as much biobehavioral reaction as pain of skin breaking procedures; 4) higher morphine exposure does not ameliorate effects of cumulative neonatal pain; 5) the combination of higher maternal stress and higher infant cortisol predicts poorer infant visual attention; 6) higher internalizing behaviors (anxiety/depressive symptoms) are evident in preterm compared to full-term toddlers as young as age 18 months, and internalizing is associated both with higher toddler basal cortisol and higher parenting stress in preterm, but not full-term toddlers. The proposed research will continue to address effects of cumulative neonatal pain/stress and analgesia on stress regulation and neurodevelopment. The overall aims are: 1) To establish whether the high cortisol levels we found in infancy persist to age 6 years in the same cohort, and whether cortisol levels are related to neurodevelopment (attention, visual memory, executive function and behavior); 2) To determine the extent to which maternal factors (parenting stress, mother-infant interaction) modulate effects of neonatal pain/stress exposure, and high infant cortisol, on neurodevelopment (attention, visual memory, executive function and behavior) at 6 years; 3) To examine whether neonatal pain/stress exposure is associated with altered brain processing and organization of visual-spatial memory at age 6 years, and whether brain processing and organization are related to attention, visual memory, executive function and behavior; 4) To determine the role of genetic variants in the serotonin transporter gene, 5-HTT, on the long term effects of early adverse experiences (neonatal pain/stress), and whether early adverse experience (neonatal pain/stress) interacts with maternal parenting stress and behaviors to alter the epigeneic regulation of the glucocorticoid receptor gene. The data from the proposed study will contribute to elucidating etiology and mechanisms of neurodevelopmental problems in children born very preterm. Further understanding of the role of maternal factors will inform strategies for intervention related to these difficulties.